Chronic early life stress induced by limited bedding and nesting (LBN) material in rodents: critical considerations of methodology, outcomes and translational potential.
The immediate and long-term effects of exposure to early life stress (ELS) have been documented in humans and animal models. Even relatively brief periods of stress during the first 10 days of life in rodents can impact later behavioral regulation and the vulnerability to develop adult pathologies, in particular an impairment of cognitive functions and neurogenesis, but also modified social, emotional, and conditioned fear responses. The development of preclinical models of ELS exposure allows the examination of mechanisms and testing of therapeutic approaches that are not possible in humans. Here, we describe limited bedding and nesting (LBN) procedures, with models that produce altered maternal behavior ranging from fragmentation of care to maltreatment of infants. The purpose of this paper is to discuss important issues related to the implementation of this chronic ELS procedure and to describe some of the most prominent endpoints and consequences, focusing on areas of convergence between laboratories. Effects on the hypothalamic-pituitary adrenal (HPA) axis, gut axis and metabolism are presented in addition to changes in cognitive and emotional functions. Interestingly, recent data have suggested a strong sex difference in some of the reported consequences of the LBN paradigm, with females being more resilient in general than males. As both the chronic and intermittent variants of the LBN procedure have profound consequences on the offspring with minimal external intervention from the investigator, this model is advantageous ecologically and has a large translational potential. In addition to the direct effect of ELS on neurodevelopmental outcomes, exposure to adverse early environments can also have intergenerational impacts on mental health and function in subsequent generation offspring. Thus, advancing our understanding of the effect of ELS on brain and behavioral development is of critical concern for the health and wellbeing of both the current population, and for generations to come.


Career plans of current orthopaedic residents with a focus on sex-based and generational differences.
An orthopaedic workforce shortage has been projected. The purpose of this study is to analyze the supply side of this shortage by ascertaining the career plans of current orthopaedic residents, comparing these plans with the career patterns of practicing orthopaedists, and identifying career-plan differences according to sex. An online, self-administered survey was e-mailed to U.S. orthopaedic residents in postgraduate year three or higher, querying them about their fellowship specialty choice and their career plans. A total of 498 residents completed the online survey; 430 respondents (86%) were male, sixty-three (13%) were female, and five (1%) did not provide information regarding sex. Ninety-one percent of the residents were planning to enroll in a fellowship, with some respondents indicating more than one subspecialty choice: 28% intended to choose sports; 21%, arthroplasty; 14%, hand surgery, 12%, trauma; 8%, pediatrics; 8%, shoulder and elbow surgery; 8%, spine surgery; 6%, foot and ankle surgery; and 2%, oncology. With regard to the top career priorities of residents in selecting a fellowship specialty, 40% indicated intellectual priorities; 36%, educational; 21%, lifestyle; and 4%, economic. Significantly more women than men were planning on pursuing a pediatric fellowship (24% versus 6%, respectively, p < 0.05) and significantly fewer were planning on pursuing a sports fellowship (11% versus 31%, respectively, p < 0.05). Significantly more women than men planned on a subspecialty-only practice (62% versus 34%, respectively, p < 0.05). The projected retirement age of sixty-four years for current residents is roughly equal to that of the previous generation. There was no difference between men and women with regard to leadership and research aspirations, projected retirement age, and projected workdays per week. However, significantly more women than men (65% versus 47%, respectively) planned on reducing their work hours or changing to part-time status at some time during their careers. There is a higher percentage of female residents (13%) than female practicing orthopaedists (4%) in the United States. We should continue efforts to collect workforce data and be proactive to avert or minimize the effect of impending orthopaedic workforce shortages on our patients. Given the trend toward an increasing proportion of female orthopaedists and the higher likelihood that they will reduce their work hours during portions of their career, policymakers should consider training more orthopaedists to ensure patient access to timely, quality orthopaedic care.


Association fibers connecting the Broca center and the lateral superior frontal gyrus: a microsurgical and tractographic anatomy.
Recently, intraoperative mapping has disclosed that, in addition to the classic language centers (that is, the Broca and Wernicke centers), other cortical regions may also play an important role in language organization. In the prefrontal cortex, although the lateral superior frontal gyrus (LSFG) could have language-related functions, there are no detailed reports that demonstrate the anatomical connection between the LSFG and other well-known language cortices, such as the Broca center. To show the existence of the structural connection, white matter association fibers between the inferior frontal gyrus (IFG) and the LSFG were examined using fiber dissection (FD) and diffusion tensor (DT) imaging-based tractography. Eight cadaveric cerebral hemispheres were dissected to reveal the association fibers between the IFG and LSFG. The DT imaging-based tractography studies targeting the prefrontal cortex were obtained in 53 right-handed patients who had no organic cerebral lesions. The association fiber tract between Brodmann area 44/45 (the Broca center in the dominant hemisphere) and LSFG were detected in all specimens by FD. In the DT imaging-based tractography studies, the tract was identified in all patients bilaterally, except for the 4 in whom the tract was detected only in the left hemisphere. This tract was spread significantly wider in the left than in the right hemisphere, and left lateralization was evident in male patients. Based on its character, this tract was named the Broca-LSFG pathway. These findings suggest a close relationship between this pathway and language organization. The structural anatomy of the Broca-LSFG pathway may explain speech disturbances induced by LSFG stimulation that are sometimes observed during intraoperative language mapping.


Molecular cloning and expression during development of the Drosophila gene for the catalytic subunit of DNA polymerase epsilon.
We have cloned the genomic DNA and cDNA of Drosophila DNA polymerase epsilon (pol-epsilon) catalytic subunit (GenBank No. AB035512). The gene is separated into four exons by three short introns, and the open reading frame consists of 6660 base pairs (bp) capable of encoding a polypeptide of 2220 amino acid residues. The calculated molecular mass is 255018, similar to that of mammalian and yeast homologues. The deduced amino acid sequence of the pol-epsilon catalytic subunit shares approximately 41% identity with human and mouse homologues as well as significant homology those of C. elegans, S. cerevisiae and S. pombe. Similar to the pol-epsilon catalytic subunits from other species, the pol-epsilon catalytic subunit contains domains for DNA polymerization and 3'-5' exonuclease in the N-terminal region, and two potential zinc-finger domains in the C-terminal regions. Interestingly, a 38 amino acid sequence in the C-terminal region from amino acid positions 1823 to 1861 is similar to the site for Mycoplasma ATP binding and/or ATPase domain (GenBank No. P47365). Northern hybridization analysis indicated that the gene is expressed at the highest levels in unfertilized eggs, followed by zero to 4h embryos and adult females, and then embryos at other embryonic stages, instar larva stages and adult males. Low levels of the mRNA were also detected at the pupa stage. This pattern of expression is similar to those of DNA replication-related enzymes such as DNA polymerase alpha and delta except for the high level of expression in adult males.


Nutritional Sustainability: Aligning Priorities in Nutrition and Public Health with Agricultural Production.
Nutrition science-based dietary advice urges changes that may have a great impact on agricultural systems. For example, the 2016 Dietary Guidelines for Americans (DGA) recommends greatly increased fruit and vegetable consumption, but the present domestic production is insufficient to accommodate large-scale adoption of these guidelines. Increasing production to the extent needed to meet the DGA will necessitate changes in an already stressed agriculture and food system and will require nutrition and agriculture professionals to come together in open and collegial discourse. All involved need to understand the stress placed on the food system by increasing populations, changing diets, and changing environments, and recognize the major diet-based public health challenges. Furthermore, there is a need to understand the intricate interplay of the myriad parts of the food system and the vast amount of work necessary to make even small changes. New systems approaches are needed, especially at the research level, where nutrition, public health, agriculture, and the food industry work together to solve interconnected problems. Future well-being depends on a sustainable food system that continues to deliver optimal health with minimal impact on the environment.


Boosting enrolment in clinical trials: validation of a regional network model.
Clinical trials of stroke therapy have been hampered by slow rates of enrolment. Our purpose is to validate a previously developed model for accelerating enrolment in clinical trials by replicating it at new locations. The model employs coordinators who travel from a host institution to enrol participants from a network of participating hospitals. Active surveillance assures identification of all eligible patients. Among 70 U.S. investigators participating in National Institutes of Health-funded trial of stroke prevention, five investigators were invited to develop local identification and outreach networks (LIONs). Each LION comprised a LION coordinating centre servicing multiple hospitals. Hospitals provided names of patients with stroke or transient ischaemic attack to researchers at the LION coordinating centre who initiated contact; patients were offered home visits for consent and randomization. Outcomes were feasibility, enrolment, data quality, and cost. Five LIONs varied in size from two to eight hospitals. All 24 hospitals we approached agreed to participate. The average monthly rate of enrolment at the research sites increased from 1.4 participants to 3.5 after expanding from a single institution model to the LION format (mean change = 2.1, range 0.9-3.7). Monthly performance improved over time. Data quality was similar for LIONs and non-LION sites, except for drug adherence which was lower at LIONs. The average cost to randomize and follow one participant during the study interval was 2.4 times the cost under the per-patient, cost-reimbursement strategy at non-LION sites. The cost ratio declined from 3.4 in year one to 1.8 in year two. The LION strategy requires unprecedented collaboration and trust among institutions. Applicability beyond stroke requires confirmation. LIONs are a practical, reproducible method to increase enrolment in trial research. Twelve months were required for the average site to reach its potential. The per-participant cost at LIONs was higher than conventional sites but declined over time.


Novel collophorina-like genera and species from Prunus trees and vineyards in Germany.
Strains with a yeast-like appearance were frequently collected in two surveys on the biodiversity of fungi in Germany, either associated with necroses in wood of Prunus trees in orchards in Saxony, Lower Saxony and Baden-Württemberg or captured in spore traps mounted on grapevine shoots in a vineyard in Rhineland-Palatinate. The morphology of the strains was reminiscent of the genus Collophorina: all strains produced aseptate conidia on integrated conidiogenous cells directly on hyphae, on discrete phialides, adelophialides and by microcyclic conidiation, while in some strains additionally endoconidia or conidia in conidiomata were observed. Blastn searches with the ITS region placed the strains in the Leotiomycetes close to Collophorina spp. Analyses based on morphological and multi-locus sequence data (LSU, ITS, EF-1α, GAPDH) revealed that the 152 isolates from wood of Prunus spp. belong to five species including C. paarla, C. africana and three new species. A further ten isolates from spore traps belonged to seven new species, of which one was isolated from Prunus wood as well. However, a comparison with both LSU and ITS sequence data of these collophorina-like species with reference sequences from further Leotiomycetes revealed the genus Collophorina to be polyphyletic and the strains to pertain to several genera within the Phacidiales. Collophorina paarla and C. euphorbiae are transferred to the newly erected genera Pallidophorina and Ramoconidiophora, respectively. The new genera Capturomyces, Variabilispora and Vexillomyces are erected to accommodate five new species isolated from spore traps. In total nine species were recognised as new to science and described as Collophorina badensis, C. germanica, C. neorubra, Capturomyces funiculosus, Ca. luteus, Tympanis inflata, Variabilispora flava, Vexillomyces palatinus and V. verruculosus.


Preparation of layered double hydroxide/chlorophyll a hybrid nano-antennae: a key step.
In the first step to obtain an efficient nano-antenna in a bottom-up approach, new hybrid materials were synthesized using a set of layered double hydroxides (LDHs) with basic properties and pure chlorophyll a (Chl a). The stability of the adsorbed monolayer of Chl a was shown to be dependent on the nature and the ratio of the different metal ions present in the LDHs tested. The hybrid materials turned out to be adequate for stabilizing Chl a on Mg/Al LDHs for more than a month under ambient conditions while a limited catalytic decomposition was observed for the Ni/Al LDHs leading to the formation of pheophytin. These changes were followed by namely XRD, DR-UV-vis and fluorescence spectroscopies of the hybrid antennae and of the solutions obtained from their lixiviation with acetone or diethylether. On Mg/Al hydrotalcites the stability of the adsorbed Chl a was equivalent for values of the metal atom ratio ranging from 2 to 4. The latter hybrids should constitute a good basis to form efficient nanoscale light harvesting units following intercalation of selected dyes. This work describes an efficient preparation of Chl a that allows scale-up as well as the obtention of a stable Chl a monolayer on the surface of various LDHs.


Identification of an atypical CD8 T cell epitope encoded by murine cytomegalovirus ORF-M54 gaining dominance after deletion of the immunodominant antiviral CD8 T cell specificities.
Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.


Analysis of the maternal components of the AIDS clinical trial group 076 zidovudine regimen in the prevention of mother-to-infant transmission of human immunodeficiency virus type 1.
To gain insight into the protective effects of the three components of the zidovudine regimen used in AIDS Clinical Trial Group (ACTG) 076 on mother-to-infant transmission of human immunodeficiency virus (HIV) type 1, 188 zidovudine-treated women and their untreated infants from five HIV-1 obstetric centers were retrospectively studied. The overall rate of mother-to-infant transmission was 12.3% (95% confidence interval [CI], 7.9%-18.0%). When the 38 women with <200 CD4 cells/microL were excluded, the mother-to-infant transmission rate was 8.8% (95% CI, 4.6%-14.8%). This rate compares favorably with the 8.3% transmission in the zidovudine arm of the ACTG 076 study. Apart from low (<200/microL) maternal CD4 cells (P = .016), no factors, including the duration of zidovudine therapy during gestation and intravenous administration of zidovudine during labor, affected the rate of mother-to-infant transmission. These findings suggest that antenatal oral zidovudine may be as effective as antenatal oral plus intravenous zidovudine during labor and the three-component ACTG 076 regimen in decreasing mother-to-infant HIV-1 transmission.


Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell-surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins.
Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). Recently, by extensive purification, it was separated from cell-lytic factor (hemolysin) and found to consist of three molecular species each with its own specificity (EGCases I, II, and III) (Ito, M., and Yamagata, T. (1989) J. Biol. Chem. 264, 9510-9519). A detergent was required for EGCases to express full activity, possibly due to their hydrophobic nature, and thus EGCases cannot be used for research on live cells. This paper presents findings on activator proteins in the culture supernatant of Rhodococcus sp. M-777 regarding the stimulation of EGCase activity in the absence of detergents. The activator protein, exhaustively purified and designated as activator II in this study, showed a single protein band on sodium dodecyl sulfate-, native-, and isoelectrofocussing-polyacrylamide slab gel electrophoresis after being stained with Coomassie Brilliant Blue. Its molecular weight and pI were 69,200 and 4.0, respectively. The activator protein enhanced the hydrolysis of glycosphingolipids in vitro and on the cell-surface by EGCase II in the absence of detergents in a concentration-dependent manner. Interestingly, activator II stimulated the activity of EGCase II much more than that of EGCase I on using asialo-GM1 as the substrate. This activator protein was found nonspecific to substrates susceptible to hydrolysis with EGCase II. Besides activator II, strain M-777 produced a second minor molecular species of activator protein designated as activator I which appeared specific for stimulating the activity of EGCase I in contrast to activator II. Following the addition of activator II, EGCase II hydrolyzed cell-surface glycosphingolipids quite efficiently at neutral pH at which hydrolysis hardly occurred at all in its absence. When using activator II in place of Triton X-100 for stimulating EGCase II activity, it was also noted to cause no damage to intact cells. It is thus possible by activator proteins to elucidate the biological functions of endogenous glycosphingolipids in situ by EGCases.


Surface Charge Properties of and Cu(II) Adsorption by Spores of the Marine Bacillus sp. Strain SG-1.
Spores of marine Bacillus sp. strain SG-1 are capable of oxidizing Mn(II) and Co(II), which results in the precipitation of Mn(III, IV) and Co(III) oxides and hydroxides on the spore surface. The spores also bind other heavy metals; however, little is known about the mechanism and capacity of this metal binding. In this study the characteristics of the spore surface and Cu(II) adsorption to this surface were investigated. The specific surface area of wet SG-1 spores was 74.7 m per g of dry weight as measured by the methylene blue adsorption method. This surface area is 11-fold greater than the surface area of dried spores, as determined with an N(2) adsorption surface area analyzer or as calculated from the spore dimensions, suggesting that the spore surface is porous. The surface exchange capacity as measured by the proton exchange method was found to be 30.6 mumol m, which is equal to a surface site density of 18.3 sites nm. The SG-1 spore surface charge characteristics were obtained from acid-base titration data. The surface charge density varied with pH, and the zero point of charge was pH 4.5. The titration curves suggest that the spore surface is dominated by negatively charged sites that are largely carboxylate groups but also phosphate groups. Copper adsorption by SG-1 spores was rapid and complete within minutes. The spores exhibited a high affinity for Cu(II). The amounts of copper adsorbed increased from negligible at pH 3 to maximum levels at pH >6. Their great surface area, site density, and affinity give SG-1 spores a high capability for binding metals on their surfaces, as demonstrated by our experiments with Cu(II).


Whole-Genome Sequencing Accurately Identifies Resistance to Extended-Spectrum β-Lactams for Major Gram-Negative Bacterial Pathogens.
There is marked interest in using DNA-based methods to detect antimicrobial resistance (AMR), with targeted polymerase chain reaction (PCR) approaches increasingly being incorporated into clinical care. Whole-genome sequencing (WGS) could offer significant advantages over targeted PCR for AMR detection, particularly for species where mutations are major drivers of AMR. Illumina MiSeq WGS and broth microdilution (BMD) assays were performed on 90 bloodstream isolates of the 4 most common gram-negative bacteria causing bloodstream infections in neutropenic patients. The WGS data, including both gene presence/absence and detection of mutations in an array of AMR-relevant genes, were used to predict resistance to 4 β-lactams commonly used in the empiric treatment of neutropenic fever. The genotypic predictions were then compared to phenotypic resistance as determined by BMD and by commercial methods during routine patient care. Of 133 putative instances of resistance to the β-lactams of interest identified by WGS, only 87 (65%) would have been detected by a typical PCR-based approach. The sensitivity, specificity, and positive and negative predictive values for WGS in predicting AMR were 0.87, 0.98, 0.97, and 0.91, respectively. Using BMD as the gold standard, our genotypic resistance prediction approach had a significantly higher positive predictive value compared to minimum inhibitory concentrations generated by commercial methods (0.97 vs 0.92; P = .025). These data demonstrate the potential feasibility of using WGS to guide antibiotic treatment decisions for patients with life-threatening infections for an array of medically important pathogens.


Shigella flexneri infection generates the lipid PI5P to alter endocytosis and prevent termination of EGFR signaling.
The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase-Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.


Dietary methionine restriction in mice elicits an adaptive cardiovascular response to hyperhomocysteinemia.
Dietary methionine restriction (MR) in rodents increased lifespan despite higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia, which are symptoms associated with increased risk for cardiovascular disease. We investigated this paradoxical effect of MR on cardiac function using young, old, and apolipoprotein E-deficient (ApoE-KO) mice. Indeed, MR animals exhibited higher heart-to-body weight ratio (w/w) and hyperhomocysteinemia with a molecular pattern consistent with cardiac stress while maintaining the integrity of cardiac structure. Baseline cardiac function, which was measured by non-invasive electrocardiography (ECG), showed that young MR mice had prolonged QRS intervals compared with control-fed (CF) mice, whereas old and ApoE-KO mice showed similar results for both groups. Following β-adrenergic challenge, responses of MR mice were either similar or attenuated compared with CF mice. Cardiac contractility, which was measured by isolated heart retrograde perfusion, was similar in both groups of old mice. Finally, the MR diet induced secretion of cardioprotective hormones, adiponectin and fibroblast growth factor 21 (FGF21), in MR mice with concomitant alterations in cardiac metabolic molecular signatures. Our findings demonstrate that MR diet does not alter cardiac function in mice despite the presence of hyperhomocysteinemia because of the adaptive responses of increased adiponectin and FGF21 levels.


Social isolation and outcomes post stroke.
To assess the relationship between social isolation and stroke outcomes in a multiethnic cohort. As part of the Northern Manhattan Stroke Study, the authors prospectively followed a cohort of patients with stroke for 5 years. Baseline data including social isolation were collected. At follow-up, the authors documented outcome events as defined by the first occurrence of myocardial infarction (MI), stroke recurrence, or death. Cox hazard models were used to calculate the hazard ratio (HR, 95% CI) for prestroke predictors of post stroke outcomes. The authors followed 655 ischemic stroke cases for a mean of 5 years. The cohort was 55% women; 17% white, 27% African American, 54% Hispanic; mean age 69 +/- 12 years. There were 265 first outcome events. In univariate analysis, coronary artery disease (OR 1.3, 1.0 to 1.7), age > 70 years (OR 1.9, 1.5 to 2.5), atrial fibrillation (AF) (OR 1.8, 1.3 to 2.5), race-ethnicity (white vs Hispanic) (OR 1.7, 1.1 to 2.9), physical inactivity (OR 1.3, 1.1 to 2.6), help at home (OR 1.8, 1.4 to 2.4), and social isolation (OR 1.4, 1.2 to 1.6) were associated with increased risk of an outcome event. No association was seen for hypertension, diabetes, education, sex, insurance, occupation, marital status, or primary care physician. In the multivariable model controlling for age, AF (OR 1.9, 1.5 to 2.5), help at home (OR 1.5, 1.1 to 2.0), and social isolation (OR 1.4, 1.1 to 1.8) predicted outcome events. Prestroke social isolation is a predictor of outcome events post stroke. Lack of social support may contribute to poorer outcomes due to poor compliance, depression, and stress.


Safety and efficacy of enzyme replacement therapy in combination with hematopoietic stem cell transplantation in Hurler syndrome.
Hurler syndrome is a debilitating genetic disease with a typical life span of 5 to 8 years. Early hematopoietic stem cell transplantation (HSCT) mitigates disease symptoms and improves survival. However, morbidity and mortality associated with HSCT can limit its success. We describe the initial experience with combined use of enzyme replacement therapy (ERT, laronidase) and HSCT in Hurler syndrome. Thirteen transplants were performed in 12 patients. ERT was given at a standard dose of 0.58 mg/kg per week. Transplant conditioning regimen and donor graft source were determined by institutional protocol. The median age at initiation of ERT was 12 months (range, 8 to 18 months). The median duration of pre-HSCT ERT was 12 weeks (range, 4 to 28). All but 1 patient tested showed decrease in urinary GAG excretion during ERT. ERT infusion-related toxicity was limited to mild reactions. Development of antibodies to laronidase did not correlate with infusion reactions or responses in urinary GAG excretion. ERT was given for a median of 7 weeks (range, 3 to 20) after HSCT. After transplantation, eight patients demonstrated complete donor engraftment and four suffered graft failure. Two patients required ventilator support and three developed acute GVHD. Eleven of the 12 patients are surviving with a median follow-up of 3 months (range, 1 to 7 months). In children with Hurler syndrome, ERT with HSCT is feasible and well tolerated. Development of antibodies against exogenous enzyme does not appear to correlate with infusion reactions or response to ERT. A prospective study is needed to determine the effect of concomitant ERT on transplant outcomes.


Patency of infrainguinal polytetrafluoroethylene bypass grafts with distal interposition vein cuffs.
Polytetrafluoroethylene (PTFE) prosthetic bypasses in the lower extremity have poor patency rates, particularly in limb salvage cases. Patency and limb salvage rates of PTFE bypasses supplemented by distal interposition vein cuffs were assessed in patients requiring revascularization for critical limb ischemia, in the absence of a suitable autologous saphenous vein. Between October 1993 and April 1996, 163 patients underwent 185 infrainguinal bypasses. Forty-three limbs in 42 patients (12 women, 30 men; mean age 67 years) did not have a suitable autologous saphenous vein (24%) and had femoropopliteal (20) and infrapopliteal (23) bypasses performed. Patients were examined prospectively at 3-month intervals during the first year and at 6-month intervals thereafter to determine graft patency and limb salvage. Postoperative anticoagulation with warfarin was used in 26 patients. Indications for operation included limb salvage in 41 extremities (21 rest pain/ulceration or gangrene, 20 rest pain alone), and disabling claudication in two. Patients were followed clinically for 2-30 months (mean 10 months). Cumulative 2-year life-table patencies for all grafts, femoropopliteal and infrapopliteal bypasses were 64%, 75% and 62%, respectively. Previous primary patencies at the authors' institution for PTFE bypasses without vein cuffs were 35%, 46% and 12% for the same categories. Cumulative life-table limb salvage for all PTFE/vein cuff bypasses in the present series was 76% compared with 37% in previous PTFE bypasses without vein cuffs. Adjunctive use of distal interposition vein cuffs improves prosthetic graft patency, while producing satisfactory limb salvage. Postoperative anticoagulation did not influence graft patency. PTFE/vein cuff for lower-extremity revascularization shows good 2-year patency and is an acceptable alternate conduit in patients with critical limb ischemia when autologous saphenous vein is absent.


Sugar, uric acid, and the etiology of diabetes and obesity.
The intake of added sugars, such as from table sugar (sucrose) and high-fructose corn syrup has increased dramatically in the last hundred years and correlates closely with the rise in obesity, metabolic syndrome, and diabetes. Fructose is a major component of added sugars and is distinct from other sugars in its ability to cause intracellular ATP depletion, nucleotide turnover, and the generation of uric acid. In this article, we revisit the hypothesis that it is this unique aspect of fructose metabolism that accounts for why fructose intake increases the risk for metabolic syndrome. Recent studies show that fructose-induced uric acid generation causes mitochondrial oxidative stress that stimulates fat accumulation independent of excessive caloric intake. These studies challenge the long-standing dogma that "a calorie is just a calorie" and suggest that the metabolic effects of food may matter as much as its energy content. The discovery that fructose-mediated generation of uric acid may have a causal role in diabetes and obesity provides new insights into pathogenesis and therapies for this important disease.


Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes.
Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N(2)-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N(2)-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N(2)-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N(2)-fixing activity is the main cause for the Sub-optimal phenotype. Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity. Effective nodules (Nodule Function 83-100 %) contained four developmental zones and N(2)-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24-57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads. Three major responses to N(2)-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N(2)-fixation of clovers.


Ethical dilemmas faced by hospice nurses when administering palliative sedation to patients with terminal cancer.
Palliative sedation is a method of symptom management frequently used in hospices to treat uncontrolled symptoms at the end of life. There is a substantial body of literature on this subject; however, there has been little research into the experiences of hospice nurses when administering palliative sedation in an attempt to manage the terminal restlessness experienced by cancer patients. Semistructured interviews were conducted with a purposive sample of seven hospice nurses who had cared for at least one patient who had undergone palliative sedation within the past year in a hospice in the south of England in the United Kingdom. A phenomenological approach and Colaizzi's stages of analysis were employed to develop themes from the data. Facilitating a "peaceful death" was the primary goal of the nurses, where through the administration of palliative sedation they sought to enable and support patients to be "comfortable," "relaxed," and "calm" at the terminal stage of their illness. Ethical dilemmas related to decision making were a factor in achieving this. These were: medication decisions, "juggling the drugs," "causing the death," sedating young people, the family "requesting" sedation, and believing that hospice is a place where death is hastened. Hospice nurses in the U.K. frequently encounter ethical and emotional dilemmas when administering palliative sedation. Making such decisions about using palliative sedation causes general discomfort for them. Undertaking this aspect of care requires confidence and competence on the part of nurses, and working within a supportive hospice team is of fundamental importance in supporting this practice.


Purification and properties of an alginate lyase from a marine bacterium.
An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.


Infrared image detail enhancement based on the gradient field specification.
Human vision is sensitive to the changes of local image details, which are actually image gradients. To enhance faint infrared image details, this article proposes a gradient field specification algorithm. First we define the image gradient field and gradient histogram. Then, by analyzing the characteristics of the gradient histogram, we construct a Gaussian function to obtain the gradient histogram specification and therefore obtain the transform gradient field. In addition, subhistogram equalization is proposed based on the histogram equalization to improve the contrast of infrared images. The experimental results show that the algorithm can effectively improve image contrast and enhance weak infrared image details and edges. As a result, it can give qualified image information for different applications of an infrared image. In addition, it can also be applied to enhance other types of images such as visible, medical, and lunar surface.


Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2.
Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.


A possible novel function of dominance behaviour in queen-less colonies of the primitively eusocial wasp Ropalidia marginata.
Unlike the queens of other primitively eusocial species, Ropalidia marginata queens are strikingly docile and non-aggressive individuals, never at the top of the behavioural dominance hierarchy of their colonies. Nevertheless, these queens are completely successful at suppressing worker reproduction, suggesting that they do not use aggression but employ some other mechanism (e.g. pheromones) to do so. Upon removal of the queen from a colony, a single worker, the 'potential queen', immediately begins to display highly elevated levels of aggression towards her nest mates. This individual becomes the next docile queen if the original queen is not returned. We attempt to understand the function of the temporary and amplified dominance behaviour displayed by the potential queen. We find that the dominance behaviour shown by the potential queen is unrelated to the number of her nest mates, their dominance ranks or ovarian condition. This suggests that aggression may not be used to actively suppress other workers and counter threat. Instead we find evidence that dominance behaviour is required for the potential queen's rapid ovarian development, facilitating her speedy establishment as the sole reproductive individual in the colony.


Safety of freeze-dried mycelia of Antrodia camphorata as a novel food pursuant to regulation (EU) 2015/2283.
Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on freeze-dried mycelia of Antrodia camphorata as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is produced by solid-state cultivation from tissue cultures derived from the fungus Antrodia camphorata. The applicant intends to market the NF in food supplements at a maximum dose of 990 mg per day. The target population is the general population. The NF mainly consists of carbohydrates, proteins and fats, and it contains numerous constituents, such as β-glucans, antroquinonol and triterpenoids. Taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. There are no concerns regarding genotoxicity of the NF. Based on a 90-day repeated dose toxicity study and a prenatal developmental toxicity study performed with the NF, the Panel derives a safe level of 16.5 mg/kg body weight per day. The Panel concludes that the NF, freeze-dried mycelia of Antrodia camphorata, is safe at the proposed use level for individuals aged 14 years and above.


Elucidating Methods for Isolation and Quantification of Exosomes: A Review.
Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.


Validity of the thin mask approximation in extreme ultraviolet mask roughness simulations.
In the case of extreme ultraviolet (EUV) lithography, modeling has shown that reflector phase roughness on the lithographic mask is a significant concern due to the image plane speckle it causes and the resulting line-edge roughness on imaged features. Modeling results have recently been used to determine the requirements for future production worthy masks yielding the extremely stringent specification of 50 pm rms roughness. Owing to the scale of the problem in terms of memory requirements, past modeling results have been based on the thin mask approximation in this application. EUV masks, however, are inherently three-dimensional (3D) in nature and thus the question arises as to the validity of the thin mask approximation. Here, we directly compare the image plane speckle calculation results using the fast two-dimensional thin mask model to rigorous finite-difference time-domain results and find the two methods to agree to within 10% in the computation of the speckle magnitude and 20% in the computation of the line-edge roughness limited depth of focus. For both types of computation, the two-dimensional method provides a conservative estimate. The 3D modeling is also used to show that layer-to-layer correlated roughness is indeed the roughness metric of most concern.


Effects of acute resistance exercise on muscle damage and perceptual measures between men who are lean and obese.
The purpose of this investigation was to assess indices of muscle damage and psychological stress between young, untrained, lean, and obese men. Using a between-subject design, 19 young men (9 lean men [age, 20.1 ± 2.1 years; body mass, 71.7 ± 5.8 kg; height, 177.8 ± 8.7 cm; body fat (BF), 14.7 ± 3.5%], 5 World Health Organization [WHO] class 1 obese men [age, 21.6 ± 2.5 years; body mass, 97.8 ± 8.6 kg; height, 176.3 ± 3.7 cm; BF, 34.7 ± 3.0%], and 5 WHO class 2 or 3 men [age, 20.0 ± 1.4 years; body mass, 120.8 ± 10.5 kg; height, 177.7 ± 5.2 cm; BF, 40.5 ± 5.8%]) volunteered and completed an acute resistance exercise (RE) protocol (6 exercises performed for 3 sets of 10 repetitions at an intensity of 85-95% of a 10 repetition maximum). Plasma myoglobin and serum creatine kinase were obtained before and immediately after exercise, and in recovery (at +110 minutes and +24 hours). Perceptual measures including rating of perceived exertion, pain and soreness, fatigue, and general soreness were assessed at different time points (during exercise for rating of perceived exertion, and for the fatigue and soreness measures before, immediately after, and at 24 hours of recovery from exercise). The primary findings of this investigation were that lean and obese, sedentary, young men do not significantly differ from each other in terms of indirect, humoral measures of muscle damage, or perceptual scales in response to a moderate-intensity acute RE bout, despite using significantly more exercise volume relative to fat mass (FM). We conclude that excess FM during daily activities of life provides a protective effect for muscle damage. When strength training individuals who are obese, practitioners should be aware of how excess FM affects muscle damage and total volume. But these considerations do not preclude individuals who are obese from using well-designed RE workouts which use free-weight, multijoint movements that stimulate all of the major muscle groups.


Waldeyer's ring lymphomas: treatment results and prognostic factors.
Optimal management of patients with localized Waldeyer's ring (WR) lymphoma remains controversial due to the lack of randomized studies and heterogenous grouping of most reported series. In this retrospective study, we have evaluated the possible prognostic factors and treatment outcome of WR non-Hodgkin's lymphoma. Between December 1993 and February 2000, 32 patients with WR lymphoma, stage I (11 patients) and stage II (21 patients) were treated. There were 17 male patients and 15 female patients with a median age of 47 years. The distribution among different anatomical sites were as follows: tonsils in 16 (50%), nasopharynx in 10 (31%), base of tongue in 6 (19%). According to Working Formulation, 10 had high-grade, 17 intermediate grade, 3 low-grade, and 2 had unclassified lymphomas. Combined chemotherapy and radiotherapy was the primary modality of therapy for intermediate or high-grade lymphoma. Radiotherapy alone was employed only in low-grade WR lymphomas. Chemotherapy was median 6 courses of CHOP (cyclophosphamide, doxorubicin (Adriamycin), vincristine, and prednisolone) in 26 patients and CEOP (cyclophosphamide, doxorubicin, etoposide, and prednisone). Radiotherapy volume was involved field and the median dose was 40 Gy. Median follow-up is 40 months (ranged from 6-82 months). Overall survival and disease-free survival (DFS) rates at 3 years are 100% and 92%, respectively. Two patients developed recurrence, both salvaged with further chemotherapy. Only one patient died because of other reasons. International Prognostic Index score (<or=2 vs. >2) is found to be an important prognostic factor for DFS. The other significant prognostic factors for DFS are performance status and serum levels of alkaline phosphatase and lactate dehydrogenase. Our results suggest that combined chemotherapy and involved field radiotherapy is appropriate treatment for stage I-II WR lymphoma. International Prognostic Index is the strongest predictor for DFS.


Contactin-2 expression in the cardiac Purkinje fiber network.
Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes. Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express beta-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca(2+) transients in Cntn2-EGFP(+) cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations. Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.


A randomized trial of the effect of soft contact lenses on myopia progression in children.
Soft contact lenses have been reported to increase the progression of myopia. The purpose of this study was to determine whether soft contact lenses affect the progression of myopia in children. Children between the ages of 8 and 11 years with -1.00 to -6.00 D myopia and less than 1.00 D astigmatism were randomly assigned to wear soft contact lenses (n = 247) or spectacles (n = 237) for 3 years. Refractive error and corneal curvatures were measured annually by cycloplegic autorefraction, and axial length was measured annually by A-scan ultrasound. Multilevel modeling was used to compare the rate of change of refractive error, corneal curvature, and axial length between spectacle and contact lens wearers. There was a statistically significant interaction between time and treatment for myopia progression (P = 0.002); the average rate of change was 0.06 D per year greater for contact lens wearers than spectacle wearers. After 3 years, the adjusted difference between contact lens wearers and spectacle wearers was not statistically significant (95% confidence interval [CI] = -0.46 to 0.02). There was no difference between the two treatment groups with respect to change in axial length (ANCOVA, P = 0.37) or change in the steepest corneal curvature (ANCOVA, P = 0.72). These data provide reassurance to eye care practitioners concerned with the phenomenon of "myopic creep." Soft contact lens wear by children does not cause a clinically relevant increase in axial length, corneal curvature, or myopia relative to spectacle lens wear. (ClinicalTrials.gov, NCT00522288.).


Shot noise limited characterization of ultraweak femtosecond pulse trains.
Ultrafast science is inherently, due to the lack of fast enough detectors and electronics, based on nonlinear interactions. Typically, however, nonlinear measurements require significant powers and often operate in a limited spectral range. Here we overcome the difficulties of ultraweak ultrafast measurements by precision time-domain localization of spectral components. We utilize this for linear self-referenced characterization of pulse trains having ∼ 1 photon per pulse, a regime in which nonlinear techniques are impractical, at a temporal resolution of ∼ 10 fs. This technique does not only set a new scale of sensitivity in ultrashort pulse characterization, but is also applicable in any spectral range from the near-infrared to the deep UV.


Protein components of mitochondrial DNA nucleoids in higher eukaryotes.
Mitochondrial DNA (mtDNA) is not packaged in nucleosomal particles, but has been reported to associate with the mitochondrial inner membrane. Gentle lysis of Xenopus oocyte mitochondria with nonionic detergent liberates a nucleoprotein complex containing mtDNA associated with a previously characterized DNA binding partner, mitochondrial transcription factor A (mtTFA), as well as a series of inner membrane proteins identified by sequencing. More extensive detergent treatment stripped most of these proteins from the DNA, leaving a limited number of proteins in a nucleoid core. Sequencing of the major proteins retained in association with mtDNA revealed the expected mtDNA binding proteins, mtTFA and mitochondrial single-stranded DNA binding protein (mtSSB), as well as four proteins not previously reported to associate with mtDNA. These include adenine nucleotide translocator 1, the lipoyl-containing E2 subunits of pyruvate dehydrogenase and branched chain alpha-ketoacid dehydrogenase and prohibitin 2. The association of several of these proteins with mtTFA-containing mtDNA nucleoids was confirmed by immunoprecipitation.


Dynamical transition in proteins and non-Gaussian behavior of low-frequency modes in self-consistent normal mode analysis.
Self-consistent normal mode analysis (SCNMA) is applied to heme c type cytochrome f to study temperature-dependent protein motion. Classical normal mode analysis assumes harmonic behavior and the protein mean-square displacement has a linear dependence on temperature. This is only consistent with low-temperature experimental results. To connect the protein vibrational motions between low and physiological temperatures, we have incorporated a fitted set of anharmonic potentials into SCNMA. In addition, quantum harmonic-oscillator theory has been used to calculate the displacement distribution for individual vibrational modes. We find that the modes involving soft bonds exhibit significant non-Gaussian dynamics at physiological temperature, which suggests that it may be the cause of the non-Gaussian behavior of the protein motions probed by elastic incoherent neutron scattering. The combined theory displays a dynamical transition caused by the softening of few "torsional" modes in the low-frequency regime ( <50 cm(-1) or <6 meV or >0.6 ps). These modes change from Gaussian to a classical distribution upon heating. Our theory provides an alternative way to understand the microscopic origin of the protein dynamical transition.


Ganglioside incorporation and release by the isolated perfused rat liver.
The fate of exogenous ganglioside GM1 labelled in the sphingosine moiety, [Sph-3H]GM1, administered as a pulse, in the isolated perfused rat liver was investigated. When a non-recirculating protocol was employed, the amount of radioactivity in the liver and perfusates was found to be dependent on the presence of BSA in the perfusion liquid and on the time elapsed after the administration of the ganglioside. When BSA was added to the perfusion liquid, less radioactivity was found in the liver and more in the perfusate at each time tested, for up to 1 h. The recovery of radioactivity in the perfusates followed a complex course which can be described by three pseudo-first-order kinetic constants. The constants, in order of decreasing velocity, are interpreted as: (a) the dilution of the labelled GM1 by the constant influx of perfusion liquid; (b) the washing off of GM1 loosely bound to the surface of liver cells; (c) the release of gangliosides from the liver. Process (b) was found to be faster in the presence of BSA, probably owing to the ability of BSA to bind gangliosides. The [Sph-3H]GM1 in the liver underwent metabolism, leading to the appearance of products of anabolic (GD1a, GD1b) and catabolic (GM2, GM3) origin; GD1a appeared before GM2 and GM3 but, at times longer than 10 min, GM2 and GM3 showed more radioactivity than GD1a. At a given time the distribution of the radioactivity in the perfusates was quite different from that of the liver. In fact, after 60 min GD1a was the only metabolite present in any amount, the other being GM3, the quantity of which was small. This indicates that the liver is able to release newly synthesized gangliosides quite specifically. When a recirculating protocol was used, there were more catabolites and less GD1a than with the non-recirculating protocol. A possible regulatory role of ganglioside re-internalization on their own metabolism in the liver is postulated.


Acceptance and Commitment Therapy, Relational Frame Theory, and the Third Wave of Behavioral and Cognitive Therapies - Republished Article.
The first wave of behavior therapy countered the excesses and scientific weakness of existing nonempirical clinical traditions through empirically studied first-order change efforts linked to behavioral principles targeting directly relevant clinical targets. The second wave was characterized by similar direct change efforts guided by social learning and cognitive principles that included cognitive in addition to behavioral and emotive targets. Various factors seem to have set the stage for a third wave, including anomalies in the current literature and philosophical changes. Acceptance and Commitment Therapy (ACT) is one of a number of new interventions from both behavioral and cognitive wings that seem to be moving the field in a different direction. ACT is explicitly contextualistic and is based on a basic experimental analysis of human language and cognition, Relational Frame Theory (RFT). RFT explains why cognitive fusion and experiential avoidance are both ubiquitous and harmful. ACT targets these processes and is producing supportive data both at the process and outcome level. The third-wave treatments are characterized by openness to older clinical traditions, a focus on second order and contextual change, an emphasis of function over form, and the construction of flexible and effective repertoires, among other features. They build on the first- and second-wave treatments, but seem to be carrying the behavior therapy tradition forward into new territory.


Two structurally different RNA molecules are bound by the spliceosomal protein U1A using the same recognition strategy.
Human U1A protein binds to hairpin II of U1 small nuclear RNA (snRNA) and, together with other proteins, forms the U1 snRNP essential in pre-mRNA splicing. U1A protein also binds to the 3' untranslated region (3'UTR) of its own pre-mRNA, inhibiting polyadenylation of the 3'end and thereby downregulating its own expression. The 3'UTR folds into an evolutionarily conserved secondary structure with two internal loops; one loop contains the sequence AUUGCAC and the other its variant AUUGUAC. The sequence AUUGCAC is also found in hairpin II of U1 snRNA; hence, U1A protein recognizes the same heptanucleotide sequence in two different structural contexts. In order to better understand the control mechanism of the polyadenylation process, we have built a model of the U1A protein-3'UTR complex based on the crystal structure of the U1A protein-hairpin II RNA complex which we determined previously. In the crystal structure of the U1A protein-hairpin II RNA complex the AUUGCAC sequence fits tightly into a groove on the surface of U1A protein. The conservation of the heptanucleotide in the 3'UTR strongly suggests that U1A protein forms identical sequence-specific contacts with the heptanucleotide sequence when complexed with the 3'UTR. The crystal structure of the hairpin II complex and the twofold symmetry in the 3'UTR RNA provide sufficient information to restrict the conformation of the 3'UTR RNA and have enabled us to build a model of the 3'UTR complex. In the U1A-3'UTR complex, sequence-specific interactions are made entirely by the conserved heptanucleotide and the last base pair (C:G) of the stem. The structure is stabilized by protein-protein contacts and by electrostatic interactions between basic amino acids of the protein and the phosphate backbone of the RNA stem regions. The formation of a protein dimer necessary for the inhibition of poly(A) polymerase requires a conformational change of the C termini of the proteins upon RNA binding. This mechanism could prevent the inhibition of poly(A) polymerase by free U1A protein. The model is consistent with biochemical data, and the protein-protein interactions within the 3'UTR complex account for the cooperativity of U1A protein binding to the 3'UTR. The model also serves as an important structural guide for designing further experiments to understand the interaction between the U1A-3'UTR complex and poly(A) polymerase.


Immunohistological evidence for a chronic intramyocardial inflammatory process in dilated cardiomyopathy.
To determine whether immunohistochemical analysis of cardiac biopsies from patients presenting clinically as dilated cardiomyopathy (DCM) show a chronic inflammatory process. Comparative case control study. Tertiary referral centre. Biopsies from 170 patients with DCM and 85 control patients with other cardiac diseases. Nine patients had sufficient interstitial inflammatory cells to be called borderline myocarditis on conventional histology, leaving 161 patients with DCM. In 78 patients with DCM (48%) there were T lymphocytes in the myocardium. In 48 (62%) of these 78 T lymphocyte densities were in the range 2-14 per high power field (HPF), equivalent to 7-50 per mm2 of tissue. In 43 (89%) interstitial and endothelial immune activation was demonstrated by MHC expression. In 30 patients with T cell counts in the range 1.5-2.0 per HPF, 80% also showed endothelial activation. Lymphocyte density correlated with increased expression of MHC class I and II antigens and the adhesion molecules ICAM, VCAM, ELAM, LFA-3, and GMP140. In all control biopsies the T lymphocyte density was less than 1.0 per HPF (less than 2-5 per mm2 of tissue). Nearly half the patients with DCM had increased T lymphocyte density and immune activation of endothelial and interstitial cells in their cardiac biopsies. A chronic autoimmune process is still active within the myocardium in a significant percentage of patients with DCM. Immunohistochemical analysis of cardiac biopsies will enhance the sensitivity of cardiac biopsy and is essential for the diagnosis of myocarditis.


Possible role of brain-derived neurotrophic factor in the pathogenesis of coronary artery disease.
The neurotrophin (NT) family, including nerve growth factor NT-3 and brain-derived neurotrophic factor (BDNF), has a critical role in the survival, growth, maintenance, and death of central and peripheral neurons. NTs and their receptors are expressed in atherosclerotic lesions; however, their significance in cardiovascular disease remains unclear. To clarify the role of NTs in the pathogenesis of coronary artery disease, NT plasma levels in the aorta, coronary sinus, and peripheral veins of patients with unstable angina (n=38), stable effort angina (n=45), and non-coronary artery disease (n=24) were examined. In addition, regional expression of BDNF in coronary arteries was examined in autopsy cases and patients with angina pectoris by directional coronary atherectomy. The difference in BDNF levels, but not NT-3, between the coronary sinus and aorta was significantly greater in the unstable angina group compared with the stable effort angina and non-coronary artery disease groups. Immunohistochemical investigations demonstrated BDNF expression in the atheromatous intima and adventitia in atherosclerotic coronary arteries. BDNF expression was enhanced in macrophages and smooth muscle cells in atherosclerotic coronary arteries. Stimulation with recombinant BDNF significantly enhanced NAD(P)H oxidase activity and the generation of reactive oxygen species in cultured human coronary artery smooth muscle cells. BDNF has an important role in atherogenesis and plaque instability via the activation of NAD(P)H oxidase.


Specificity of octopine uptake by Rhizobium and pseudomonas strains.
The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.


Colony and Single Cell Level Analysis of the Heterogeneous Response of Cryptococcus neoformans to Fluconazole.
Cryptococcus neoformans is a human fungal pathogen that can cause fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug administered to treat cryptococcosis. When exposed to the inhibitory concentration of FLC, C. neoformans exhibits heteroresistance where a small subpopulation of cells develops into FLC-resistant colonies. FLC-resistant cells are aneuploids with regard to specific beneficial chromosomal regions. Factors underlying the potential for only certain C. neoformans cells in a genetically isogenic population to become FLC-resistant are unknown. In this study, we systematically examine the heterogeneous response of C. neoformans to FLC at a colony and individual cell level. We find that the heterogeneity in response to FLC is reflected by variable diminishment of the ergosterol at the plasma membrane. A population of C. neoformans spread on a semi-solid medium displays two types of outcomes following FLC exposure. The first outcome is colonies consisting of non-resistant cells (survivors). The size of colonies consisting of survivors ranges from a few cells to visible colonies, which reflects intrinsic phenotypic heterogeneity of the C. neoformans population. The second outcome is FLC-resistant cells forming colonies of sizes significantly larger as compared to colonies made of survivors. We propose a model that describes how a distribution of these types of cellular responses within a population changes depending on FLC concentration and factors that influence the rate of cellular growth including temperature, media type, growth phase, and the age of cells. Our findings highlight a complex nature of the response to a fungistatic drug and provide insights that may help to optimize FLC therapy.


Effects of combining the fungi Beauveria bassiana and Metarhizium anisopliae on the mortality of the tick Amblyomma variegatum (ixodidae) in relation to seasonal changes.
The effect of formulation, fungal concentration, type and seasonal changes on the mortality of the tick Amblyomma variegatum was investigated. A previous study demonstrated high pathogenicity of strains of the fungi Beauveria bassiana and Metarhizium anisopliae against the ticks Rhipicephalus appendiculatus and Amblyomma variegatum (Kaaya et al. J Invertebr Pathol 1996; 67: 15-20). The present study was undertaken to explore possible additive or synergistic effects of the two fungi on A. variegatum. The effects of oil and water formulations at different concentrations of each fungus and combination of the two on the mortality of A. variegatum in the laboratory and in the field during the wet and dry seasons were determined and compared. The oil formulation performed better in all assays, with highest tick mortality of 92% occurring during the wet season at conidia concentration of 1 x 10(10) conidia/ml of the mixed fungi compared to 49% for the water formulation at similar conidia concentration. However, at the same conidial concentration during the dry season, mortalities in the field were relatively low with the mixture of the fungi recording 24% and 17% tick mortality for the oil and water formulation respectively. The effect of infecting the ticks with a cocktail of the two fungi was inconclusive under more controlled conditions in the laboratory but field results under both wet and dry seasons indicated significant differences between the separate and mixed fungi infections. The results demonstrate a potential of cocktail formulations in the control of ticks and possibly of other arthropod pests.


Stabilization and Validation of 3D Object Position Using Multimodal Sensor Fusion and Semantic Segmentation.
The stabilization and validation process of the measured position of objects is an important step for high-level perception functions and for the correct processing of sensory data. The goal of this process is to detect and handle inconsistencies between different sensor measurements, which result from the perception system. The aggregation of the detections from different sensors consists in the combination of the sensorial data in one common reference frame for each identified object, leading to the creation of a super-sensor. The result of the data aggregation may end up with errors such as false detections, misplaced object cuboids or an incorrect number of objects in the scene. The stabilization and validation process is focused on mitigating these problems. The current paper proposes four contributions for solving the stabilization and validation task, for autonomous vehicles, using the following sensors: trifocal camera, fisheye camera, long-range RADAR (Radio detection and ranging), and 4-layer and 16-layer LIDARs (Light Detection and Ranging). We propose two original data association methods used in the sensor fusion and tracking processes. The first data association algorithm is created for tracking LIDAR objects and combines multiple appearance and motion features in order to exploit the available information for road objects. The second novel data association algorithm is designed for trifocal camera objects and has the objective of finding measurement correspondences to sensor fused objects such that the super-sensor data are enriched by adding the semantic class information. The implemented trifocal object association solution uses a novel polar association scheme combined with a decision tree to find the best hypothesis-measurement correlations. Another contribution we propose for stabilizing object position and unpredictable behavior of road objects, provided by multiple types of complementary sensors, is the use of a fusion approach based on the Unscented Kalman Filter and a single-layer perceptron. The last novel contribution is related to the validation of the 3D object position, which is solved using a fuzzy logic technique combined with a semantic segmentation image. The proposed algorithms have a real-time performance, achieving a cumulative running time of 90 ms, and have been evaluated using ground truth data extracted from a high-precision GPS (global positioning system) with 2 cm accuracy, obtaining an average error of 0.8 m.


SU-E-T-593: VMAT Vs. IMRT for Treatment of Neoplasms of the Brain: Dosimetric Quality and Delivery.
A retrospective study was performed to determine any dosimetric or delivery benefits for treatment planning with intensity-modulated radiotherapy (IMRT) versus volumetric-modulated arc therapy (VMAT) for the treatment of brain neoplasms. Eighteen patients treated with modulated brain radiotherapy treatments were included in this study (primary treatment volumes of 15.3 to 374.9 cc). IMRT and VMAT plans were generated for each patient using the same criteria for prescription coverage and normal tissue sparing. IMRT optimizations ranged from five to seven fields and VMAT from two to four arcs. Plans were generated with Varian Eclipse treatment planning system utilizing AAA-8615 dose calculation algorithm. VMAT optimizations provided limited dosimetric advantages versus IMRT. VMAT provided superior treatment volume coverage (volumes receiving 95%, 100% of prescription dose (V95%, V100%)) over IMRT, but differences were not statistically significant (paired t-test p > 0.05). Relative maximum dose values, conformity and homogeneity indices also exhibited no statistical differences. IMRT plans resulted in similar mean brain minus treatment volume (Brain- TV) dose (mean = 1460.1 vs. 1506.3 cGy; p = 0.056). The volume of Brain- TV receiving 40Gy was lower for VMAT than IMRT (average = 38.96 vs. 44.97 cc; p = 0.050). Maximum skin dose was lower for VMAT (mean = 4568.8 vs. 5063.3cGy; p = 0.006), as well as skin V20Gy (6.56% vs 7.69%; p = 0.027) and V40Gy (0.56% vs. 0.35%; p = 0.017). VMAT plans required fewer fields along with fewer MU than IMRT (mean = 388.2 vs. 721.1 MU, respectively), allowing for approximately 20% faster delivery times. VMAT treatments significantly reduced treatment time due to reduced MU and fewer fields. Certain skin and Brain-TV high dose spread parameters were superior for VMAT as compared to IMRT plans. All other dosimetric parameters tested were statistically equivalent for VMAT and IMRT techniques.


The functional demands on the intact limb during walking for active trans-femoral and trans-tibial amputees.
The aim of this study was to investigate the loading demands placed on the intact limb in terms of joint moments and power for active trans-femoral and trans-tibial amputees in comparison to a group of able-bodied subjects. Four (4) trans-tibial, 4 trans-femoral amputees and 10 able-bodied subjects walked at 1.2m.s(-1) along a walkway whilst kinematic data from both the intact and prosthetic limbs, and kinetic data from the intact limb only were collected. A Panasonic VHS video camera was used to film subjects walking in the sagittal plane with simultaneous force data collected from a Kistler force platform. The amputees were found to compensate for the functional loss of one or more joints by increasing net joint moments and power output on their intact limb compared to able-bodied subjects. At the intact limb ankle, the range of motion, peak dorsiflexor moment and power generation at toe-off increased. At the intact limb knee, power generation during stance and extensor moments and power absorption at toe-off increased. At the intact limb hip, extensor moment and power absorption during stance, and hip flexor moment and power generation at toe-off increased. These findings were partly attributed to the prostheses used but mainly to adaptation mechanisms displayed by trans-femoral and trans-tibial amputees. They have implications for the mobility of amputees and the long term health of their joints. It was recommended that prosthesis design, prosthesis fitting and training in the use of the prosthesis were all factors which could be investigated with a view to minimising intact limb loading.


Amplitude modulation and apodization of quasiphase-matched interactions.
We propose several techniques to modulate the local amplitude of quasi-phase-matched (QPM) interactions in periodically poled lithium niobate waveguides and demonstrate apodization by using each of these techniques. When the hard edges are removed in the spatial profile of the nonlinear coupling, the sidelobes of the frequency tuning curves are suppressed by 13 dB or more, compared with a uniform grating, consistent with theoretical predictions. The sidelobe-suppressed gratings are useful for frequency conversion devices in optical communication systems to minimize interchannel cross talk, while the amplitude modulation techniques in general have potential uses in applications that require altering the tuning curve shapes.


ATR kinase inhibitor AZD6738 potentiates CD8+ T cell-dependent antitumor activity following radiation.
DNA-damaging chemotherapy and radiation therapy are integrated into the treatment paradigm of the majority of cancer patients. Recently, immunotherapy that targets the immunosuppressive interaction between programmed death 1 (PD-1) and its ligand PD-L1 has been approved for malignancies including non-small cell lung cancer, melanoma, and head and neck squamous cell carcinoma. ATR is a DNA damage-signaling kinase activated at damaged replication forks, and ATR kinase inhibitors potentiate the cytotoxicity of DNA-damaging chemotherapies. We show here that the ATR kinase inhibitor AZD6738 combines with conformal radiation therapy to attenuate radiation-induced CD8+ T cell exhaustion and potentiate CD8+ T cell activity in mouse models of Kras-mutant cancer. Mechanistically, AZD6738 blocks radiation-induced PD-L1 upregulation on tumor cells and dramatically decreases the number of tumor-infiltrating Tregs. Remarkably, AZD6738 combines with conformal radiation therapy to generate immunologic memory in complete responder mice. Our work raises the possibility that a single pharmacologic agent may enhance the cytotoxic effects of radiation while concurrently potentiating radiation-induced antitumor immune responses.


[Providing insight into the construct decisional conflict and its usability to assess shared decision making].
The increased attention for shared decision making (sdm) in mental health care creates a need to evaluate its application. The construct decisional conflict, which refers to the satisfaction of patients regarding both the decision making process and the decisions made, could be of added value.
 AIM: Clarifying decisional conflict and reflecting on its feasibility to evaluate sdm in mental health care.
 METHOD: A literature study exploring the construct of decisional conflict was conducted, followed by a translation of the results into a visual model.
 RESULTS: Decisional conflict is a multi-dimensional construct and consists of factors influencing the decision making process (information, support, values clarity), level of uncertainty concerning the options and the quality of the decision making. Decisional conflict can be illustrated by using a model and assessed with the Decisional Conflict Scale.
 CONCLUSION: Decisional conflict is informative and useful in the evaluation of the application of sdm and improvement of the quality of the decision making in mental health care as well. This is of importance since patients who experienced less decisional conflict are more engaged in treatment and show better clinical outcomes.


Association of circulating adhesion molecules with lung function. The CARDIA study.
Systemic inflammation has been associated with reduced lung function. Adhesion molecules, such as intercellular adhesion molecule (ICAM)-1 and P-selectin, figure importantly in initiating the inflammatory response. We studied the association between ICAM-1 and P-selectin concentrations and lung function in the Coronary Artery Risk Development in Young Adults study. Spirometry testing was conducted at years 5, 10, and 20. ICAM-1 and P-selectin were assayed at year 15. Complete data were obtained from 2,455 participants. We first predicted year-20 lung function from year-15 ICAM-1 concentration data. After controlling for race, gender, height, age, physical activity, smoking status, alcohol intake, BMI, and asthma status, all taken at year 15, the year-20 FVC was 164 mL higher (p < 0.0001) and FEV(1) was 164 mL higher (p = 0.0003) in the lowest ICAM-1 concentration quartile than the highest ICAM-1 quartile, whereas the FEV(1)/FVC ratio showed no association (p = 0.25). We then predicted the year-15 ICAM-1 concentration from year-5 lung function and change in lung function (year 10 - year 5). The year-15 ICAM-1 concentration was about 13 ng/mL higher in the lowest vs highest quartile of either the year-5 FVC (p = 0.01) or year-5 FEV(1) (p = 0.005). Year-15 ICAM-1 concentration was unrelated to year-5 FEV(1)/FVC ratio. Greater loss in FVC and FEV(1) (year 10 - year 5) also was associated with higher year-15 ICAM-1 concentrations. Associations between P-selectin and lung function followed a similar but weaker pattern to that observed for ICAM-1. These data suggest a bidirectional association between circulating adhesion molecules, such as ICAM-1 and P-selectin, and pattern of lung function change in adults.


Autoantibodies to a 64-kilodalton islet cell protein precede the onset of spontaneous diabetes in the BB rat.
Spontaneous insulin-dependent diabetes mellitus (IDDM) in the BB rat is associated with the presence of antibodies to a 64-kilodalton rat islet cell protein. These protein antibodies appeared in young animals and remained for as long as 8 weeks before the clinical onset of IDDM. Antibodies to a 64-kilodalton human islet cell protein were found to be associated with human IDDM. Detection of the antibodies may therefore be used to predict an early immune reaction against pancreatic B cells.


Whole genome sequencing identifies allelic ratio distortion in sperm involving genes related to spermatogenesis in a swine model.
Transmission Ratio Distortion (TRD), the uneven transmission of an allele from a parent to its offspring, can be caused by allelic differences affecting gametogenesis, fertilization or embryogenesis. However, TRD remains vaguely studied at a genomic scale. We sequenced the diploid and haploid genomes of three boars from leukocytes and spermatozoa at 50x to shed light into the genetic basis of spermatogenesis-caused Allelic Ratio Distortion (ARD). We first developed a Binomial model to identify ARD by simultaneously analysing all three males. This led to the identification of 55 ARD SNPs, most of which were animal-specific. We then evaluated ARD individually within each pig by a Fisher's exact test and identified two shared genes (TOP3A and UNC5B) and four shared genomic regions harbouring distinct ARD SNPs in the three boars. The shared genomic regions contained candidate genes with functions related to spermatogenesis including AK7, ARID4B, BDKRB2, GSK3B, NID1, NSMCE1, PALB2, VRK1 and ZC3H13. Using the Fisher's test, we also identified 378 genes containing variants with protein damaging potential in at least one boar, a high proportion of which, including FAM120B, TDRD15, JAM2 or AOX4 among others, are associated to spermatogenesis. Overall, our results show that sperm is subjected to ARD with variants associated to a wide variety of genes involved in different stages of spermatogenesis.


Laparoscopic gastric gastrointestinal stromal tumor resection: the mayo clinic experience.
Laparoscopic resection of gastric gastrointestinal stromal tumors (GISTs) is safe and effective. Retrospective medical record review. Tertiary referral center. Patients undergoing laparoscopic resection of gastric GISTs from April 1, 2000, to April 1, 2006. Demographic data, diagnostic workup, operative technique, tumor characteristics, morbidity, mortality, and follow-up. Thirty-three patients underwent attempted laparoscopic resection of gastric GISTs, with 31 operations completed laparoscopically. The mean patient age was 68 years (age range, 35-86 years). The female to male ratio was 18:15. Sixteen patients (49%) were asymptomatic, and their tumors were found incidentally. Of 24 patients (73%) who underwent preoperative endoscopic ultrasonography, the results of fine-needle aspiration verified the diagnosis in 13 patients (54%). The mean operative time was 124 minutes (range, 30-253 minutes). A combined endoscopic-laparoscopic approach was used in 11 patients (33%). The mean tumor size was 3.9 cm (range, 0.5-10.5 cm). Two patients (6%) underwent conversion to an open procedure. The median hospital stay duration was 3 days. The mean follow-up was 13 months (range, 3-64 months). There were no local recurrences. Three patients (9%) experienced complications, including 1 wound infection and 2 episodes of upper gastrointestinal tract bleeding. There were no mortalities. Although technically demanding, the laparoscopic approach to gastric GISTs is safe and effective, resulting in a short hospital stay duration and low morbidity.


Mode of regulation of the extracellular signal-regulated kinases in the pancreatic beta-cell line MIN6 and their implication in the regulation of insulin gene transcription.
Physiological concentrations of glucose that lead to Ca2+ entry and insulin secretion activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the MIN6 pancreatic beta-cell line. Here we show that this activation is inhibited by the down-regulation of protein kinase C (PKC) and by genistein, an inhibitor of protein tyrosine kinases. In contrast with results obtained in other cell types, neither the epidermal growth factor activity nor the Src family protein tyrosine kinases seem to be involved in the Ca2+-dependent activation of ERKs. inhibition of tyrosine phosphatases by vanadate leads to the activation of ERKs. As observed in the response to glucose, this activation is dependent on Ca2+ entry through L-type voltage-dependent Ca2+ channels. Thus the activation of ERKs in response to glucose depends on PKC and possibly on a tyrosine kinase/tyrosine phosphatase couple. To define the role of ERK activation by glucose we studied the regulation of transcription of the insulin gene. We found that this transcription is regulated in the MIN6 cells in the same range of glucose concentration as in primary islets, and that specific inhibition of mitogen-activated protein kinase kinase, the direct activator of ERK, impaired the response of the insulin gene to glucose. This was observed by analysis of the transfected rat insulin I gene promoter activity and a Northern blot of endogenous insulin mRNA.


Oxyradical generation from leukocytes during endotoxin-induced microcirculatory disturbance in rat mesentery--attenuating effect of cetraxate.
By using the intravital microscope equipped with digital imaging processor, we investigated the granulocyte-mediated oxidative burst during the endotoxin-induced microvascular derangement in rat mesentery. The leukocyte behavior after the injection of acridine orange was detected by using a silicon-intensified target camera, the erythrocyte velocity was measured by using a high-speed video camera system, and the luminol-dependent photoemission was visualized by an ultrasensitive photon-counting camera in lipopolysaccharide (LPS)-treated microvascular beds. At 60 min after the LPS administration, a significant leakage of FITC-labeled albumin was observed along mesenteric venules under a fluorescence microscopy. The number of sticking leukocytes increased in association with the decrease in erythrocyte velocity after starting the LPS infusion. The luminol-dependent chemiluminescence in microvascular beds gradually increased over that recorded prior to LPS exposure and was fourfold higher 60 min after the start of LPS infusion. The distribution of the photoemission clearly corresponded to the venular endothelium, to which leukocytes adhered. In blood samples taken from the mesenteric vein at 60 min after the LPS administration, a decrease in the number of granulocytes and increases of total and individual chemiluminescence activities were observed. These results suggest that LPS induces oxidative burst from granulocytes on the venular endothelium. Cetraxate, an inhibitor of proteases including plasmin, significantly inhibited the leukocyte activation and prevented alterations in microvascular hemodynamics induced by LPS in vivo, whereas it had no effect on the LPS-induced oxyradical generation from adherent leukocytes in vitro. The present study demonstrates that proteases such as plasmin may play an important role in the pathogenesis of endotoxin-induced microvascular disturbances.


Minimal clinically important improvement and patient acceptable symptom state for subjective outcome measures in rheumatic disorders.
The concepts of minimal clinically important improvement (MCII) and patient acceptable symptomatic state (PASS) could help in interpreting results of trials involving patient-reported outcomes by translating the response at the group level (change in mean scores) into more clinically meaningful information by addressing the patient level as "therapeutic success (yes/no)." The aims of the special interest group (SIG) at OMERACT 8 were to discuss specific issues concerning the MCII and PASS concepts, especially the wording of the external anchor questions used to determine the MCII and PASS estimates, and to move toward a consensus for the cutoff values to use as the MCII and PASS in the different outcome criteria. The purpose of this SIG at OMERACT 8 was to inform participants of the MCII and PASS concepts and to agree on MCII and PASS values for pain, patient global assessment, and functional impairment.


Genetic Analysis of High Protein Content in 'AC Proteus' Related Soybean Populations Using SSR, SNP, DArT and DArTseq Markers.
Key message: Several AC Proteus derived genomic regions (QTLs, SNPs) have been identified which may prove useful for further development of high yielding high protein cultivars and allele-specific marker developments. High seed protein content is a trait which is typically difficult to introgress into soybean without an accompanying reduction in seed yield. In a previous study, 'AC Proteus' was used as a high protein source and was found to produce populations that did not exhibit the typical association between high protein and low yield. Five high x low protein RIL populations and a high x high protein RIL population were evaluated by either quantitative trait locus (QTL) analysis or bulk segregant analyses (BSA) following phenotyping in the field. QTL analysis in one population using SSR, DArT and DArTseq markers found two QTLs for seed protein content on chromosomes 15 and 20. The BSA analyses suggested multiple genomic regions are involved with high protein content across the five populations, including the two previously mentioned QTLs. In an alternative approach to identify high protein genes, pedigree analysis identified SNPs for which the allele associated with high protein was retained in seven high protein descendants of AC Proteus on chromosomes 2, 17 and 18. Aside from the two identified QTLs (five genomic regions in total considering the two with highly elevated test statistic, but below the statistical threshold and the one with epistatic interactions) which were some distance from Meta-QTL regions and which were also supported by our BSA analysis within five populations. These high protein regions may prove useful for further development of high yielding high protein cultivars.


Delay of adjuvant chemotherapy initiation following breast cancer surgery among elderly women.
Delay in the diagnosis of breast cancer is associated with worse stage distribution at diagnosis and decreased survival. However, the occurrence of delay in the delivery of adjuvant therapy and its impact on prognosis is not well understood. To investigate the timeliness of initiation of adjuvant chemotherapy following surgery for breast cancer, we used data from the Surveillance, Epidemiology, and End-Results (SEER)-Medicare database. Among women > or = 65 years diagnosed between 1992 and 1999 with stages I-II breast cancer, we used linear regression and Cox proportional hazards models to investigate the time intervals between surgery and initiation of adjuvant chemotherapy, factors associated with delay, and the effect of delay on survival. Our sample consisted of 5003 women who received adjuvant chemotherapy. Of these, 47% initiated chemotherapy within 1 month, 37% between 1 and 2 months, 6% between 2 and 3 months and 10% >3 months (delay) following surgery. Delay was associated with increasing age, residing in a rural location, being unmarried, earlier tumor stage, hormone receptor positivity, mastectomy, and non-receipt of radiation therapy. Survival did not differ among patients who initiated chemotherapy within 1, 2, or 3 months after surgery. Delay beyond 3 months was, however, associated with increased disease-specific mortality (HR 1.69; 95% CI 1.31-2.19) and overall mortality (HR 1.46; 95% CI 1.21-1.75). Among older patients, moderate delays in the receipt of adjuvant chemotherapy occur frequently, but long delays (>3 months) are uncommon. While early initiation of therapy is no benefit, significant delays are associated with increased mortality. Whether this reflects the medical impact of the delay of chemotherapy or factors associated with delay is unclear, but until this is clarified, patients should be encouraged to initiate treatment without significant delay.


Population pharmacokinetics of ciprofloxacin in neonates and young infants less than three months of age.
Ciprofloxacin is used in neonates with suspected or documented Gram-negative serious infections. Currently, its use is off-label partly because of lack of pharmacokinetic studies. Within the FP7 EU project TINN (Treat Infection in NeoNates), our aim was to evaluate the population pharmacokinetics of ciprofloxacin in neonates and young infants <3 months of age and define the appropriate dose in order to optimize ciprofloxacin treatment in this vulnerable population. Blood samples were collected from neonates treated with ciprofloxacin and concentrations were quantified by high-pressure liquid chromatography-mass spectrometry. Population pharmacokinetic analysis was performed using NONMEM software. The data from 60 newborn infants (postmenstrual age [PMA] range, 24.9 to 47.9 weeks) were available for population pharmacokinetic analysis. A two-compartment model with first-order elimination showed the best fit with the data. A covariate analysis identified that gestational age, postnatal age, current weight, serum creatinine concentration, and use of inotropes had a significant impact on ciprofloxacin pharmacokinetics. Monte Carlo simulation demonstrated that 90% of hypothetical newborns with a PMA of <34 weeks treated with 7.5 mg/kg twice daily and 84% of newborns with a PMA ≥34 weeks and young infants receiving 12.5 mg/kg twice daily would reach the AUC/MIC target of 125, using the standard EUCAST MIC susceptibility breakpoint of 0.5 mg/liter. The associated risks of overdose for the proposed dosing regimen were <8%. The population pharmacokinetics of ciprofloxacin was evaluated in neonates and young infants <3 months old, and a dosing regimen was established based on simulation.